The goals of this project application are two-fold. First, this project aims to advance the principal investigator's scientific training and career development as a post-doctoral fellow in the laboratory of Professor Michael Gilmore at the Massachusetts Eye and Ear Infirmary. Second, this project aims to investigate the molecular biology of a unique toxin, called cytolysin, which is produced by Enterococcus faecalis bacteria. With the advent of antibiotic therapy, E. faecalis has transformed from a prominent member of the commensal gut microbiota into a multi-drug resistant, hospital-acquired pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin called cytolysin, which lyses both bacterial and eukaryotic cells in response to quorum signals. Originally described in the 1930s, the cytolysin is a member of a large class of lanthionine-containing bacteriocins produced by Gram-positive bacteria. While the cytolysin shares some core features with other lantibiotics, it possesses unique characteristics as well. Importantly, little is currently known about how expression of the individual genes within the cytolysin operon is controlled, as well as the precise function of the final gene in the operon, the cytolysin immunity factor cylI. The long-ter objectives of this research project are to better understand the mechanisms of cytolysin expression and immunity, in order to better treat and control highly virulent, hospital-acquired E. faecalis infections. The first aim of this project is to: 1. Establish how the cytolysin operon is regulated transcriptionally. To carry out this aim, RNA sequencing (RNAseq) will be conducted in order to: map the transcription start sites of all cytolysin operon transcripts, identify antisese and processed mRNA transcripts that may regulate the cytolysin operon, and identify genes that show differential expression in bacterial strains that do or do not harbor the cytolysin operon, and between the uninduced and induced states of cytolysin expression. The second aim of this project is to: 2. Characterize the product of the immunity gene cylI, and determine how immunity to cytolysin is transferred between cells. To carry out this aim, the function and substrate preferences for the CylI protein will be determined using both in vitro and whole cell assays. Additionally, a cytolysin transfer assay will be developed in order to determine the timing, conditions, and efficiency of cytolysin operon transfer. Overall, this project will advance understanding of the basic biological mechanisms of an important human pathogen, while simultaneously providing an enriching and worthwhile experience to the principal investigator.